This competitive renewal will continue an investigation of the effects of simulated ischemia and reperfusion on isolated adult ventricular cardiomyocytes. In addition, it will investigate myocytes from ventricular muscle subjected to ischemia and reperfusion in vivo. Because there is a wealth of data on in vivo myocardial ischemia and reperfusion in the dog, and because canine hearts and ventricular cardiomyocytes resemble those from humans, this competitive renewal will focus on canine ventricular cardiomyocytes. It will also investigate human ventricular cardiomyocytes when feasible. Three hypotheses will be tested: 1) That the mitochondrial inner membrane permeability transition, or "pore" formation, is activated by simulated myocardial ischemia/reperfusion in isolated canine myocytes in vitro and by ischemia/reperfusion in vivo, and that the mitochondrial inner membrane permeability transition contributes to cell injury. Attempts will be made to inhibit the transition with cyclosporin A and other agents such as phospholipase inhibitors that work synergistically with cyclosporin A to preserve the mitochondrial inner membrane permeability barrier. Cyclosporin analogues, which unlike cyclosporin A do not inhibit calcineurin, yet still inhibit the inner membrane permeability transition, will be investigated as well; 2) That the ability of the sarcoplasmic reticulum to accumulate and release Ca2+ is altered by ischemia/reperfusion and that altered reactions of the sarcoplasmic reticulum contribute to post-ischemic contractile dysfunction. These studies will employ digitonin- permeabilized myocytes to assess the functional competence of the sarcoplasmic reticulum and to probe the status of the Ca2+ efflux pathways; 3) That ischemia/reperfusion produces alterations in the alpha and beta adrenergic receptors that have a negative impact on excitation- contraction coupling. These lead to post-ischemic contractile dysfunction and contribute to the development of ventricular arrhythmias. Receptor numbers and subtypes as well as second messenger production will be assessed in suspensions of intact canine myocytes. Effects on excitation- contraction coupling will be assessed in field stimulated single canine cells superfused with selected adrenergic agonists and antagonists. Excitation-contraction will be assessed with video-edge monitoring of cell length and fura-2 fluorescence microscopy to monitor intracellular free [Ca2+]. The above hypotheses focus on systems capable of influencing and being influenced by Ca2+ overload during ischemia and reperfusion. The resulting specific aims to test these hypotheses are logical extensions of work accomplished during the previous funding period.